Project Summary Auditory hair cells connect to afferent nerve fibers through ribbon synapses, where auditory signals are converted from graded membrane potentials in hair cells into spikes in afferent nerve fibers. In vivo studies conducted in adult animals have shown that in response to sound afferent fibers fire spikes with remarkable temporal precision, but the underlying synaptic mechanisms are still poorly understood. A major hurdle is the lack of a fully mature model system for dual patch-clamp analysis in vitro. The afferent fiber terminals in the adult mammalian cochlea are small and mostly inaccessible to patch-clamp analysis, and results from the immature cochlea are insufficient to explain in vivo findings. Here we propose to examine auditory transmission in the amphibian papilla of adult bullfrogs, which is a unique model system where spiking patterns of afferent fibers in vivo have been recapitulated with paired patch-clamp experiments in vitro. Using this model system, we will combine dual patch-clamp recording, glutamate uncaging and computer modeling to address a central and fundamental question in hearing: how small and fast-changing signals in auditory hair cells are transmitted to afferent fibers precisely and tirelessly. First, we will examine the desensitization properties of AMPA receptors in afferent fiber terminals, and determine how these properties allow the ribbon synapses to maintain synaptic strength under continuous glutamate release from hair cells. Second, we will demonstrate that Ca2+ influx caused by pre-depolarization leads to rapid priming of synaptic vesicles and makes them releasable in response to brief voltage changes. We will test the hypothesis that this priming of synaptic vesicles is mediated by a second Ca2+ sensor with biophysical properties distinct from the one for synaptic vesicle fusion. Lastly, we will elucidate the cellular mechanisms for multivesicular release, a hallmark of hair cell ribbon synapses. We will test three major candidate mechanisms: fusion pore flickering, nanodomain coupling of synaptic vesicles and Ca2+ channels, and coordination of synaptic ribbons. The results obtained through this project will answer several long-standing questions of inner ear physiology, and expand our knowledge of the fundamental rules governing synaptic transmission at auditory hair cell synapses.